The outer membrane protein complex (OMPC) of Neisseria meninioitidis is a very effective immunogenic carrier of polysaccharide antigens and has been approved by regulatory agencies worldwide as a vaccine component (PedvaxHIB.RTM.). OMPC is a potential immunogenic carrier for peptide antigens, especially those derived from the V3 domain of gp 120, an envelope protein of the human immunodeficiency virus. The principal neutralization determinant of this V3 domain has been extensively mapped [Emini, E. A. and Conley, A. J. (1992), The Development of an AIDS Vaccine: Progress and Perspectives, in Challenges of the 1990's. New Diseases and New Therapies: Acquired Immunodeficiency Syndrome (AIDS) (R. P. Luthy and R. G. Douglas, eds.) Hanley and Belfus, Philadelphia; 85-91 and references cited therein] and has been shown to reside in the V3 disulfide loop. Indeed, substantial antipeptide responses are observed for such OMPC-peptide conjugates. A common feature of these peptides is the presence of a Gly Pro Gly Arg (Seq. Id:1:) sequence.
These peptide conjugates were prepared by reacting an electrophilic derivative of the peptide with a thiol derivative of OMPC, which was obtained by reacting the protein with N-acetylhomocysteine thiolactone [Marburg, S., et al., J. Am. Chem. Soc. 108, 5282-5287], as outlined in Scheme 1: ##STR1##
This thiolactone has been used to introduce a thiol functionality into small molecules and macromolecules for a variety of purposes outlined in table 1, and has been particularly useful because it unambiguously defines covalency by a `bigeneric spacer` concept, [Marburg et al., J.A.C.S. 108, 5258-5287(1986); U.S. Pat. No. 4, 695, 624]. The bigeneric spacer technology is especially significant in production of conjugate vaccines, because hydrolysis of the conjugate allows for quantitation of specific spacer components. Thus, one commonly produced spacer is S-(carboxymethyl)homocysteine, SCMHC, while another is S-(carboxymethyl)cysteamine, SCMCA. Both of these spacer components show up in a "window" in an amino acid analysis of a hydrolyzed conjugate where no naturally occurring amino acids migrate, and are thus readily quantified. This is an important safety feature because in quantitating the SCMHC or SCMCA products, the number of epitopes appended to the carrier can be determined.
TABLE 1 ______________________________________ Application of N-Acetyl Homocysteine Thiolactone as a thiolating agent Molecule type thiolated Utility ______________________________________ aminoglycoside protein conjugation muramyl dipeptide polysaccharide conjugation HSA drug conjugation enzymes polystyrene conjugation albumin microspheres peptide-protein conjugation cell surface carbohydrate cell surface labeling lactoglobulin increased heat stability oligonucleotide fluorescence labelling amino lipid liposome preparation ______________________________________
Frequently, however, OMPC-peptide conjugate preparations using N-acetylhomocysteine thiolactone technology are characterized by very poor protein recoveries (&lt;15%) due to irreversible precipitation of the conjugate. This hampers use of such conjugates as a vaccine material and necessitates development of a practical solution to this problem. Examination of the peptide sequences that resulted in precipitation upon conjugation revealed that all were positively charged in that they contained one or more of the basic amino acids arginine, histidine and lysine, and none of the acidic ones, such as glutamic and aspartic acids. This is in contrast to polysaccharide conjugates which are almost always acidic polymers that form anions at neutral pHs and do not suffer the precipitation problems observed with the peptide conjugates (see U.S. Pat. No. 4, 695, 624).
The present invention provides acylated derivatives of homocysteine thiolactone incorporating acidic functionalities. Use of these novel compounds in lieu of the N-acetyl derivative in the conjugation generates linking molecules which impart one to two negative charges per ligand in the conjugate at neutral pHs.
Klotz, I. M. and Heiney, R. E. [J. Am. Chem. Soc. 81, 3802-3803, (1959)]reported the use of S-acetylmercaptosuccinic anhydride to thiolate proteins. However, the product of that work did not result in production of an easily quantifiable spacer (mercaptosuccinyl), and only introduced a single negative charge.
Use of the novel compounds of the instant invention in conjugate formation has been found to ameliorate the problems of conjugate insolubility, particularly when cationic ligands are used, while at the same time providing easily quantifiable linker degradation products.